Clinical Significance And Regulatory Mechanism Of CD133Expression During Carcinogenesis Of Colorectal Cancer

Colorectal cancer is one of the most common malignant tumors. It is the third leading cause of cancer-related morbidity and the second leading cause of mortality, with an estimated incidence of1million new cases and a mortality of>500000deaths annually in the world. In recent years, the incidence of colorectal cancer has dramatically increased in China. Although much improvement has been achieved in the treatment protocols, metastasis and/or recurrence always cause the failure of therapy and death of patients.Recently, the heterogeneity of tumors was revealed. Only a minor subpopulation of tumor cells were found in malignant tissues named cancer stem cells (CSCs) or tumor initiating cancer cells (TICs), which could reinitiate the original tumor and maintain the heterogeneity of tumor tissues in animal models. CSCs were first found in acute myeloid leukemia, and subsequently in most solid tumors, such as breast cancer, glioblastoma, melanoma, lung cancer, prostate cancer and colorectal cancer. CSCs resided in GO cell cycle and inherently resistanted to cytotoxic effect of chemotherapy or radiation therapy. All the properties of CSCs contribute to the metastasis or recurrence of tumors. CSCs expressed many stem cell related genes, such as Nanog、 SHH、NOTCH、HES、oct-4/3and some biomarkers, in which CD133is one of the most important molecules.CD133(prominin-1), a five-transmembrane glycoprotein, with a molecular weight of120kDa, was initially described as a marker specific for CD34+human hematopoietic progenitor cells (HSCs), normal stem cells of the neural, epithelial, and endothelial lineages. Resent research suggested that CD133was one of the most important cancer stem cell markers. It was over expressed in various solid tumors, including retinoblastoma, teratocarcinoma, leukaemia, glioblastoma, colorectal carcinoma and gastric carcinoma. Two groups firstly indentified CD133+colorectal tumor cells as colorectal tumor stem cells. The studies demonstrated that CD133expression was dramaticly increased in colorectal carcinoma compared to normal colorectal tissues, and CD133+cells could generate new tumor in NOD/SCID mouse. Later studies proved that CD133expression was correlated with clinical parameters of colorectal cancer patients, such as clinical stage, TNM stage, distant metastasis, lymphoid metastasis. Additionally, it was also found as an independent prognostic marker for overall survival of patients.Complicated regulatory mechanism of CD133expression has also been found. Studies demonstrated that abnormal methylation in colorectal tumor cells can inhibited the expression of CD133. While another study proved aberrant demethylation in advanced colorectal tumors could upregulate CD133expression. Some other researchers found the expression of CD133was also regulated by mTOR signal and hypoxia-inducible factor1-α (HIF1-α). Recently, Kristel Kemper et al reported the mutation of Ras-Raf signal pathway could result in up-regulated expression of CD133. Moreover, Ute Bissels’study on hematopoietic stem cells demonstrated that microRNA142-3p regulated the expression of CD133during the differentiation of HSCs. This result indicated that CD133expression can be post-transcriptionally regulated.MicroRNAs (miRNAs) are important posttranscriptional regulators that are-22nucleotides in length. Colon carcinogenesis represents a stepwise progression from benign polyps to invasive adenocarcinomas and distant metastasis. It is believed that these pathologic changes are contributed by aberrant activation or inactivation of protein-coding proto-oncogenes and tumor suppressor genes. However, recent discoveries in microRNAs research have reshaped our understanding of the role of non-protein-coding genes in carcinogenesis of colorectal tumor.This study focused on colorectal carcinoma, one of the most common malignant. Firstly, the expression of CD133was analyzed in primary colorectal cancer tissues, matched non-tumor tissues, or benign polyp tissues by immunohistochemistry (IHC), and clinical significance of CD133expression in colorectal cancer was analyzed. Furthermore, other stem cell related molecules were also investigated during carcinogenesis of colorectal cancer. At last, the regulatary mechanism of aberrant CD133expression in colorectal cancer tissues was explored. Based on these studies, we endeavored to clarify the abnormal activation of stem cells during carcinogenesis of colorectal cancer, and provide new idea for research and clinical therapy of colorectal cancer.Part I Clinical Significance and Dynamic Expressions of CD133during Carcinogenesis of Colorectal CancerObjective:To investigate expression CD133in primary colorectal cancer tissues, matched non-tumor tissues, or benign polyp tissues. Explore clinical significance of CD133expression in colorectal cancer.Methods:158primary colorectal cancer tissues and matched non-tumor tissues,71benign polyp tissues were recruitment and the expression of CD133were determined by immunohistochemistry. Fresh colorectal cancer tissues and matched non-tumor tissues from8patients underwent curative surgical resection, fresh benign polyp tissues from8patients excised under endoscope were mechanically and enzymatically disaggregated into single-cell suspensions. The expression of CD133on particular cells was investigated by flow cytometry analysis (FCM).Results:(1) Immunohistochemistry results demonstrated that CD133was typically expressed on cellular membrane in colorectal cancer tissues, matched non-tumor tissues, or benign polyp tissues with a positive percentage of45.57%(72/158),5.06%(8/158),15.49%(11/71) respectively. Significant difference was found in three type tissues (P=0.001).(2) Flow cytometer analysis demonstrated that CD133positive cells in colorectal cancer tissues, matched non-tumor tissues, or benign polyp tissues was significantly different (1.51±0.67%、3.48±1.66%、12.61±6.84%respectively)(p=0.001). These further confirmed immunohistochemistry results.(3) The expression of CD133positively correlated with histological grade (p=0.033), lymph node metastasis (p=0.001), distant metastasis (p=0.001) and TNM stage (p=0.001). CD133positive cases had a shorter median survival time (36.0months) than CD133negative cases (71.0months)(p=0.001).Conclusion:CD133expression was step-wise increased during carcinogenesis of colorectal cancer and positively correlated with histological grade, lymph node metastasis, distant metastasis and TNM stage of colorectal cancer. CD133expression was also independent prognostic marker for overall survival of patients.Part II Dynamic Expressions of Stem Cell Related Molecules During Carcinogenesis of Colorectal CancerObjective:To investigate the expression of CD44, another important biomarker for CSCs, and stem cell related genes in primary colorectal cancer tissues, matched non-tumor tissues, or benign polyp tissues.Methods:158primary colorectal cancer tissues and matched non-tumor tissues,71benign polyp tissues were recruitment and the expression of CD44, oct-4were determined by immunohistochemistry. Fresh colorectal cancer tissues and matched non-tumor tissues from33patients underwent curative surgical resection, fresh benign polyp tissues from33patients excised under endoscope were collected and transcription of stem cell related genes oct-4, klf-4, Sox2, Nanog and c-myc were determined by Real-time PCR. Meanwhile single-cell suspensions were prepared by mechanically and enzymatically disaggregation and expression of oct-4was investigated by flow cytometry analysisResults:By immunohistochemistry, CD44expression was detected on cell membrane and extracellular matrix in colorectal cancer tissues, matched non-tumor tissues, or benign polyp tissues. The expression was significant different in three type tissues and positively correlated with dynamic expressions of CD133during carcinogenesis of colorectal cancer.(2) Among stem cell related genes, oct-4, not klf-4, Sox2, Nanog neither c-myc was found step-wise increased in transcription level.(3) Immunohistochemistry results demonstrated that oct-4expressed on nucleoid and the expression was step-wise up-regulated correlating with CD133expression. Flow cytometer detections demonstrated that expression of oct-4was significantly different in normal, benign or malignant tissues (1.40±0.78%、2.91±1.57%、11.37±6.32%respectively).Conclusion:In this study, up-regulated expression of CD44was identified during carcinogenesis of colorectal cancer. Stem cell related gene, oct-4was also found step-wise up-regulated in normal, benign, or malignant tissues, both in transcription level and protein level. Our results indicated that compared to benign tissues, not only phenotype changes, but also aberrant biological activation of stem cells occurred in malignant tissues. These could contribute to carcinogenesis and development of colorectal cancer.Part Ⅲ Post-Transcriptional Regulatory Function of microRNA378on CD133ExpressionObjective:To explore potential post-transcriptional regulatory microRNAs on CD133expression. Dual luciferase assay was performed to confirm post-transcriptional regulatory function of particular microRNAs.Methods:Fresh colorectal cancer tissues and matched non-tumor tissues from33patients underwent curative surgical resection, fresh benign polyp tissues from33patients excised under endoscope were collected and transcription of CD133was determined by Real-time PCR. MicroRNA array was conducted to analyse different expression of microRNAs among normal, benign, malignant colorectal tissues. Bioinformatics was performed to predict potential microRNA which may regulate CD133expression. Dual luciferase reporter plasmids were constructed and the regulatory function of microRNA was confirmed by luciferase assay. Expression of CD133on HCT116transfected with microRNA was analyzed by FCM to further confirm regulatory function of particular microRNA.Results:(1) Conflicted with prontein level, the transcriptional level of CD133had no significant difference in normal, benign and malignant colorectal tissues (p>0.05).(2) By microRNA array analysis,8microRNAs were found significantly decreased in colorectal cancer compared with normal colorectal tissues.(3) By bioinformatics prediction, microRNA378was found have potential binding site with CD1333’UTR.(4) Dual luciferase reporter plasmid containing whole length of CD1333’UTR was successfully constructed. Significant inhibitional effect of microRNA378or microRNA142-3p on luciferase translation of reporter plasmid was detected by luciferase assay.(5) Furthermore, dual luciferase reporter plasmid containing wild type (w378/PGL3) or mutant (m378/PGL3) microRNA378binding site were successfully constructed. Luciferase assay demonstrated that luciferase translation of w378/PGL3, not m378/PGL3was inhibited by microRNA378transfection.(6) Expression of CD133on HCT116was significantly down regulated by transfected with microRNA378or microRNA142-3p in vitro.Conclusion:There was no significant difference in transcription of CD133in normal, benign or malignant colorectal. CD133expression was regulated by microRNA378. MicroRNA378expression inhibited during carcinogenesis of colorectal cancer, which may contributed to aberrant expression of CD133.In summary, this study has successfully accomplished the investigations as follows:(1) CD133expression step-wise increased during carcinogenesis of colorectal cancer and positively correlated with histological grade, lymph node metastasis, distant metastasis and TNM stage. CD133expression was also independent prognostic marker for overall survival (2) CD44dynamic expression was identified concordance with CD133during carcinogenesis of colorectal cancer. Stem cell related gene, oct-4transcription and translation was also found step-wise up-regulated in normal, benign, or malignant tissues. Compared with benign tissues, not only phenotype alteration, but also aberrant biological activation of stem cells occurred in malignant tissues. These could contribute to carcinogenesis and development of colorectal cancer.(3) There was no significant difference of transcription of CD133in normal, benign or malignant tissues. CD133expression was regulated by microRNA378. Expression of microRNA378was inhibited during carcinogenesis of colorectal cancer, which may contribute to aberrant expression of CD133.